HAX-1 was identified ~10 years ago as a binding partner of HS1, a protein involved in the maturation of T-cells. The presence of two Bcl Homology (BH) domains in its NH2-terminus suggested that it might play key roles in mediating cell survival. Using in vitro and in vivo systems, we and others have demonstrated that HAX-1 promotes cell survival by inhibiting the activation of initiator caspase-9 and death caspase-3, and by contributing to the regulation of Ca2+ homeostasis, through its direct interaction with the SarcoEndoplasmic Reticulum Ca2+ ATPase (SERCA) pump and its regulator phospholamban (PLN). Importantly, these previous studies have solely focused on variant 1, the prototypical HAX-1 protein that is abundantly expressed in several species. Recent evidence, however, has indicated that the HAX-1 gene is heavily spliced, giving rise to a number of isoforms with distinct molecular compositions and possibly functional activities. In view of these observations, our laboratory set forth to examine the presence and properties of HAX-1 variants in rat myocardium. Using RT-PCR analysis and 2D gel electrophoresis, we confirmed the presence of at least seven HAX-1 isoforms (variant I-variant VII). Transient transfections of variants (v) vI-vVII in cultures of rat cardiac H9C2 cells combined with subcellular fractionation indicated that they encode proteins with molecular masses of ~35-20 kDa, that target to both the mitochondrial and SR membranes. Variants I, V, VI and VII exerted significant anti-apoptotic activity following induction of apoptosis with different stimuli, whereas variants II, III and IV exacerbated cell death, with vIV being the most potent. Although overexpression of vIV per se in H9C2 cardiocytes did not affect their viability, it markedly enhanced cell death in response to an apoptotic insult. Taken together, our findings indicate that HAX-1 comprises a subfamily of proteins residing in the mitochondrial and SR membranes that may promote either cell survival or cell death. We therefore hypothesize that HAX-1 proteins may act antagonistically to regulate cardiomyocyte survival in response to an apoptotic stimulus. To keep our studies focused, we plan to examine the properties of the pro-apoptotic vIV in relation to the anti-apoptotic vI, which display the most potent pro-/anti-apoptotic activities. Consequently, we will investigate the mechanistic pathways through which HAX-1 vIV may oppose the anti-apoptotic activity of vI, using a combination of molecular, cellular and biochemical methodologies (Aim 1), and examine if in vivo down-regulation of vIV through adeno associated viral mediated RNA interference, may restore cardiac morphology and function in rats subjected to pressure overload through transverse aortic constriction (Aim 2). The proposed studies will significantly extent our current knowledge on the diverse activities of the HAX-1 subfamily of proteins in regulating cell survival and cell death, and their potential use as novel, therapeutic targets for cardiac disease. PUBLIC HEALTH RELEVANCE: Heart failure (HF) remains a leading cause of mortality in the developing world. Current drug treatment has limited efficiency, and in advanced HF, left ventricular assist devices or heart transplantation are the ultimate options. In the last 10 years, molecular and cellular interventions have been explored including gene and cell therapy approaches. Our studies will provide important information about the roles of a novel subfamily of proteins, namely HAX-1, in regulating cardiomyocyte survival and death, and their potential use as therapeutic targets for cardiac disease.